Enterohemorrhagic and enteropathogenic E. coli (EHEC and EPEC) are leading causes of bacterial-mediated diarrhea and are responsible for the annual death of several hundred thousand people. The applicant's long-term goal is to characterize the biological activities of a novel group of EHEC virulence proteins and to design new therapeutic strategies for E. coli disease. The objective of this particular application is to determine the mechanism of action of the EHEC NleF protein. The central hypothesis for the proposed research is that NleF is a translocated effector protein that contributes to EHEC virulence by disrupting the host response to EHEC infection. This hypothesis is based on the observations that i) deletion of nleF from a pathogen closely related to EHEC results in attenuated survival during an in vivo infection, and ii) NleF interacts specifically with the yeast exocyst, a protein complex that directs secretory vesicles to distinct sites on the plasma membrane. The specific aims are to: [unreadable] 1. Characterize the cellular localization of NleF in the infected intestinal epithelium. The hypothesis is that NleF localizes to host secretory vesicles where it disrupts their proper trafficking and interaction with the host plasma membrane. To test this hypothesis, cell biology experiments will be conducted to characterize the subcellular structures to which NleF associates following its translocation into infected host cells. [unreadable] 2. Identify the host binding partners of NleF. The hypothesis is that NleF interacts with the exocyst, a protein complex that directs secretory vesicles to the plasma membrane. To test this hypothesis, a yeast two-hybrid screen, immunoprecipitation experiments, and affinity chromatography will be conducted to determine the host cell-binding partners of NleF. [unreadable] 3. Characterize the role of NleF in disease. The hypothesis is that NleF is an essential virulence factor that disrupts the host response to EHEC infection. To test this hypothesis, the ability of an EHEC strain containing an isogenic deletion of nleF to cause disease in a germ-free piglet infection model of human EHEC disease will be measured. [unreadable] Lay Summary: E. coli has emerged as a significant source of food borne disease, often from ingestion of under-cooked beef products (hamburger disease). A new group of E. coli proteins has been discovered. These proteins are likely to contribute to diarrhea by disrupting the ability of intestinal cells to secrete proteins involved in immunologic defense The applicant is characterizing the biological activities of these proteins with the goal of developing new therapeutic strategies for E. coli disease. [unreadable] [unreadable] [unreadable]